The proposed research is a continued study of the involvement of the Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), and other proteins in the splicing of group I introns. Group I introns use RNA catalyzed splicing reactions, but require proteins for efficient splicing in vivo, presumably to facilitate correct RNA folding. We identified three nuclear genes, cyt-18, cyt-19, and cyt-4, that encode trans-acting components required for splicing group I introns in Neurospora mitochondria. During the current grant period, we showed that the CYT-18 protein, the mt TyrRS, is itself sufficient to splice group I introns in vitro, that it recognizes highly conserved structural features of the group I intron catalytic core, and that it stabilizes the core in a conformation required for catalytic activity. We also obtained evidence that CYT-18 can substitute for an RNA structure, P5a,b,c, required for the self-splicing of the Tetrahymena rRNA intron, and we shoed that CYT-18 could promote reverse splicing under physiologically relevant conditions and thus potentially contribute to intron transposition. the cyt-19 component contributes to efficient splicing in vivo, probably by helping fold the precursor RNA, whereas the CYT-4 protein has significant similarity to gene products involved in cell cycle protein phosphatase functions and may be regulatory. The findings for CYT-18 suggest that the group I intron catalytic core may have structural features that resemble those in tRNAs, which could reflect convergent evolution or an evolutionary relationship between group I introns and tRNAs. The findings for CYT-4 raise the possibility that RNA catalyzed splicing reactions are regulated by protein phosphorylation. Specific aims are: (1) To continue to investigate the function of the CYT-18 protein and its interaction with the intron RNA and tRNATyr, using biochemical and genetic approaches. We are particularly interested in how CYT-18 stabilizes the active structure of the intron core, the extent of structural similarity between the group I intron core and tRNAs, and whether CYT-18 uses similar interactions to bind group I introns and tRNATyr. These studies would include a collaboration with Dr. Thomas Steitz (Yale) to determine the structures of CYT-18/RNA complexes. (2) To investigate the evolution and consequences of protein-assisted splicing of group I introns. Initial objectives are to elucidate the structural basis for the lack of self-splicing of CYT-18-dependent group I introns in Neurospora mitochondria and to investigate how the TyrRS adapts to function in splicing. Longer term objectives are to investigate the adaptation of other proteins to function in splicing, to analyze in detail the functional equivalence of CYT-18 and the P5a,b,c structure of the Tetrahymena intron, and to establish a system for investigating intron transposition via CYT-18-dependent reverse splicing in E. coli. (3) To identify the cyt-19 component, determine its role in splicing, and if it is the target of a CYT-4-mediated regulatory pathway involving protein phosphorylation. The research is intended to provide novel information about the interaction of catalytically active RNAs with proteins required for catalytic activity and about the evolution of introns and splicing mechanisms, which are fundamentally important in eukaryotes.